Impact of waterpipe and tobacco cigarette smoking on global DNA methylation and nuclear proteins genes transcription in spermatozoa: a comparative investigation.

BACKGROUND: Waterpipe smoking is harmful and dangerous, and it is a growing threat to public health. OBJECTIVES: This study was performed to evaluate the influence of waterpipe smoking on global DNA methylation, DNA fragmentation, and protamine deficiency in spermatozoa compared to cigarette heavy smokers and nonsmokers, and to determine whether the transcription levels of spermatozoa nuclear proteins genes 'PRM1, PRM2, and H2BFWT' in waterpipe smokers are different compared to cigarette heavy smokers and nonsmokers. METHODS: A total of 900 semen samples were collected from males with a mean age of 32.5 ± 6.3 years (300 waterpipe smokers, 300 cigarette heavy smokers, and 300 nonsmokers). The nucleic acids were isolated from purified spermatozoa, and then the global DNA methylation and transcription levels of the PRM1, PRM2, and H2BFWT genes were assessed using ELISA and qPCR, respectively. RESULTS: A significant increase was found in the level of global DNA methylation (8.6 ± 0.6 ng/µl vs. 7.1 ± 0.6 ng/µl and 4.7 ± 0.6 ng/µl, p < 0.001), protamine deficiency (72.8 ± 15.3 vs. 51.7 ± 19.2 and 15.3 ± 5.9%, p < 0.001), and DNA fragmentation (73.4 ± 13.4 vs. 50.5 ± 18.9 and 9.3 ± 4.3%, p < 0.001) in waterpipe smokers compared to cigarette heavy smokers and nonsmokers. A significant increase was shown in the transcription levels of PRM1, PRM2, and H2BFWT genes in waterpipe smokers compared to cigarette heavy smokers and nonsmokers (p < 0.001). A down-regulation was found in the transcription level of these genes in different smoker groups compared to nonsmokers (<0.001). CONCLUSION: This study suggests that waterpipe smoking is more harmful than cigarette smoking on semen parameters, global DNA methylation, and transcription of nuclear protein genes.

Effect of hubble-bubble smoking on global DNA methylation and transcription levels of protamine and histone genes in human spermatozoa.

This study was conducted to assess the impact of hubble-bubble smoking on global DNA methylation, DNA fragmentation; protamine deficiency of spermatozoa, and to determine whether the transcription levels of the protamine and histone genes are different in hubble-bubble smokers compared to nonsmokers. Five hundred semen samples were collected from males with an average age of 32.2 ± 6.1 years (300 hubble-bubble smokers "60%" and 200 nonsmokers "40%"). The nucleic acid was isolated from purified sperm, then ELISA and qPCR were used to evaluate the global DNA methylation and transcription level of protamine and histone, respectively. A significant elevation in global DNA methylation, protamine deficiency, and DNA fragmentation was found in hubble-bubble smokers compared to nonsmokers (P < 0.0001). A significant decline was shown in transcription levels of protamine and histone genes in hubble-bubble compared to nonsmokers (P < 0.0001). Additionally, a down-regulation in the transcription levels of protamine and histone was revealed in hubble-bubble compared to nonsmokers with fold change (0.0001 and 0.007, respectively). In conclusion, this study provided proof that hubble-bubble smoking has a negative impact on global DNA methylation, DNA fragmentation, protamine deficiency, and the transcription of protamine and histone genes in spermatozoa, and these findings influence negatively males' fecundity.

Investigation of the Predictive Factors of Diminished Ovarian Reserve in Women Aged Less Than 40 Years and Undergoing ICSI Cycle.

Diminished ovarian reserve (DOR) is one of the primary causes of poor ICSI outcomes. Therefore, this study was performed to speculate which of the following parameters: AMH, AFC, and women's age can be used as a predictor factor of the DOR in women aged < 40 years. This prospective study enrolled 500 women suffering from idiopathic infertility problems and who underwent GnRH antagonist multiple-dose stimulation protocol. The women were divided into two groups: normal fertility (FSH ≤ 10 mIU/mL, n = 300) and DOR (FSH > 10 mIU/mL, n = 200). At the time of the study, the average of women age was 29.3 ± 5.7 years. A significant reduction was found in AMH level, AFC, number of mature, immature oocytes, fertilized oocytes, embryos transferred, and ß-hCG level in the DOR group compared to the normal fertility group (P < 0.001). Conversely, a significant increase was shown in the age of the DOR group compared to the normal fertility group (30.8 ± 5.8 vs. 28.2 ± 5.4, respectively; P < 0.001). A significant negative association was found between the AFC, the number of mature oocytes, fertilized oocytes, embryos transferred, and the basal level of FSH in the DOR group (P < 0.01). The receiver operating characteristics (ROC) demonstrated that AMH level and AFC had the highest accuracy, followed by age in the prediction of DOR (P < 0.001) with a cut-off value of ≤ 1.2 ng/mL, ≤ 4.5, and > 29.5 years, respectively. This study exhibited that the levels of AMH and AFC are the best biomarkers, followed by age for the prediction of DOR in women < 40 years old. Furthermore, AMH is the only independent factor that is significantly related to DOR in women.

Cigarette heavy smoking alters DNA methylation patterns and gene transcription levels in humans spermatozoa.

Tobacco smoking is considered the most common reason of death and infertility around the world. This study was designed to assess the impact of tobacco heavy smoking on sperm DNA methylation patterns and to determine whether the transcription level of ALDH3B2, PTGIR, PRICKLE2, and ALS2CR12 genes is different in heavy smokers compared to non-smokers. As a screening study, the 450 K array was used to assess the alteration in DNA methylation patterns between heavy smokers (n = 15) and non-smokers (n = 15). Then, four CpGs that have the highest difference in methylation level (cg16338278, cg08408433, cg05799088, and cg07227024) were selected for validation using deep bisulfite sequencing in an independent cohort of heavy smokers (n = 200) and non-smokers (n = 100). A significant variation was found between heavy smokers and non-smokers in the methylation level at all CpGs within the PRICKLE2 and ALS2CR12 gene amplicon (P < 0.001). Similarly, a significant variation was found in the methylation level at nine out of thirteen CpGs within the ALDH3B2 gene amplicon (P < 0.01). Additionally, eighteen CpGs out of the twenty-six within the PTGIR gene amplicon have a significant difference in the methylation level between heavy smokers and non-smokers (P < 0.01). The study showed a significant difference in sperm global DNA methylation, chromatin non-condensation, and DNA fragmentation (P < 0.001) between heavy smokers and non-smokers. A significant decline was shown in the transcription level of ALDH3B2, PTGIR, PRICKLE2, and ALS2CR12 genes (P < 0.001) in heavy smokers. In conclusion, heavy smoking influences DNA methylation at several CpGs, sperm global DNA methylation, and transcription level of the PRICKLE2, ALS2CR12, ALDH3B2, and PTGIR genes, which affects negatively the semen parameters of heavy smokers.

Serum leptin in diabetic nephropathy male patients from Gaza Strip.

OBJECTIVE: To assess serum leptin in diabetic nephropathy male patients from Gaza Strip. MATERIALS AND METHODS: This case-control study comprised 132 type 2 diabetic patients and 44 non-diabetic controls. The diabetic patients were classified into three groups; 44 normoalbuminurics, 44 microalbuminurics and 44 macroalbuminurics. Data were obtained from questionnaire interview, and biochemical analysis of blood and urine samples. Patients and controls were matched for age and body mass index (BMI). RESULTS: Serum leptin was significantly higher in micro- and macro-albuminuric patients (14.6 ±â€¯11.7 and 15.6 ±â€¯13.5 ng/ml) than controls and normoalbuminurics (5.9 ±â€¯4.0 and 8.1 ±â€¯7.6 ng/ml) with P < 0.05. In general, serum glucose, urea, createnine, cholesterol, triglycerides, low density lipoprotein cholesterol (LDL-C), urinary albumin and albumin creatinine ratio (ACR) were increased in diabetic groups compared to non-diabetics, and reaching their maximum increase in macroalbuminurics whereas high density lipoprotein cholesterol (HDL-C), urinary creatinine and glomerular filtration rate (GFR) were decreased reaching its maximum decrease in macroalbuminurics. Serum leptin showed significant positive correlations with diabetes duration (r = 0.188, P = 0.020), glucose (r = 0.298, P < 0.001), cholesterol (r = 0.323, P < 0.001), triglycerides (r = 0.361, P < 0.001), LDL-C (r = 0.248, P = 0.001) and urinary albumin (r = 0.256, P = 0.001) whereas negative significant correlations were found with HDL-C (r = -0.313, P < 0.001) and urinary creatinine (r = -0.202, P = 0.007). CONCLUSION: The comitant raise of serum leptin with urinary albumin combined with decrease in GFR makes leptin eligible candidate as a biomarker for progression towards diabetic nephropathy in type 2 diabetes.

Serum vitamin D status in type 2 diabetic patients from Gaza Strip.

OBJECTIVE: To assess serum vitamin D status and its relations to other biochemical parameters in type 2 diabetic patients from Gaza Strip. MATERIALS AND METHODS: This case-control study included 58 type 2 diabetic patients as well as 58 non-diabetic controls. Patients and controls were matched for age and gender. Data were obtained from questionnaire interview, and biochemical analysis of blood samples. RESULTS: Serum vitamin D was significantly lower in diabetic patients compared to non-diabetic controls (25.9 ±â€¯11.0 versus 34.6 ±â€¯13.8 ng/dl, % difference = 28.8%, P < 0.001). The number of patients having vitamin D deficient, insufficient and sufficient were 6 (10.4%), 35 (60.3%) and 17 (29.3%) compared to controls of 3 (5.2%), 16 (27.6%) and 39 (67.2%), respectively (χ2 = 14.672, P < 0.001). Serum glucose, glycated hemoglobin (HbA1c), serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and triglycerides were significantly higher in patients than in controls whereas serum insulin, high density lipoprotein cholesterol (HDL-C) and calcium were significantly lower in patients. Serum vitamin D showed significant negative correlations with HbA1c (r = - 0.186, P = 0.046), ALT (r = - 192, P = 0.040) and AST (r = - 0.188, P = 0.044) whereas significant positive correlations were found with HDL-C (r = 0.188, P = 0.044) and calcium (r = 0.239, P = 0.010). CONCLUSION: The significant negative and positive correlations of vitamin D with HbA1c and calcium, respectively suggests that vitamin D supplementation would be of potential therapeutic value in clinical settings for controlling of type 2 diabetes and more importantly its complications. However, a well-designed clinical trials are needed to define the contribution of vitamin D status and therapy in the global diabetes problem.

Childhood lead poisoning in Gaza Strip, the Palestinian Authority.

PURPOSE: To assess lead poisoning among a pediatric population in Gaza Strip, the Palestinian Authority. METHODS: A total of 1705 questionnaires and blood samples were collected from children aged 2-6 years, by the finger stick capillary procedure, for the assessment of blood lead level (BLL), using the LeadCare kits. The samples were collected from children living close to lead processing units (hotspots) and far 100-500 m away (general population). Management of elevated BLL was achieved by gavage of chelating agent d-penicillamine at a dose of 30 mg kg-1 body weight/day for two weeks for children having BLL above 20 µg/dl. Data were statistically analyzed using SPSS computer program version 22. RESULTS: Distribution of children in Gaza Strip by sampling process illustrated those 326 children (19.1%) living in lead processing units (hot spots) and 1379 children (80.9%) from location far away 100-500 m from host spot (general population). The mean BLL was 10.4 µg/dl. A total of 440 children (25.8%) were found to be having BLL ≥ 10 µg/dl while 1265 children (74.2%) have BLL < 10 µg/dl. BLL < 10 µg/dl was taken as a cut point due to CDC standards. The prevalence of BLL in hotspot areas in children who were exposed to lead and have BLL ≥ 10 µg/dl was 95.7% while in general population it was 9.3%. The difference between the study population was statistically significant (p < 0.01). Statistical significant differences between the study populations were found among several independent variables of risk factors such as household location and exposure sources, and occupational exposure. Gavage of d-penicillamine significantly reduced BLL to the acceptable level (BLL < 10 µg/dl). CONCLUSIONS: Childhood lead poisoning accounts for a substantial burden in Gaza Strip. Hotspots of lead-related industries are the high risk areas that contributing to high BLLs in children. d-Penicillamine was effective in the treatment of lead poisoning among children. Health education and treatment campaigns should be designed to minimize or prevent childhood lead poisoning in Gaza Strip.

The clinical effects of CYP2C19 *2 allele frequency on Palestinian patients receiving clopidogrel after percutaneous coronary intervention.

BackgroundCYP2C19 loss-of-function polymorphic alleles (*2 and *3) have been documented to impair clopidogrel metabolism, and represent a risk factor for major adverse cardiac events. CYP2C19 polymorphism exhibits marked ethnic heterogeneity. Objective To determine the prevalence of CYP2C19 *2 and *3 alleles in a cohort of Palestinian patients managed with percutaneous coronary intervention and dual antiplatelet therapy, and to determine their role in causing major adverse cardiac events. Setting The blood samples were collected at the European Gaza Hospital, and the molecular techniques performed at the molecular genetics laboratory of the Islamic university of Gaza. Method The frequency of CYP2C19 *2 and *3 alleles was determined in 110 patients managed with percutaneous coronary intervention and clopidogrel. Genotyping was performed by PCR-RFLP. Personal and clinical data was obtained from patient record and 6-month follow-up for major adverse cardiac events. Main outcome measureCYP2C19 genotype, personal and clinical data and incidence of major adverse cardiac events. Results The frequency of CYP2C19 *1, *2 and *3 alleles was 82.3%, 15.5% and 2.3% respectively. Genotyping analysis showed that, 67.3% were homozygotes for CYP2C19 *1, 27.3% were *1/*2, 2.7% with *1/*3 genotype, 1.8% were *2/*3 and 0.9% were *2/*2. These frequencies were consistent with those of Caucasian populations. According to this study the poor metabolizers phenotype frequency was 2.7%, which is in the same range reported in Caucasians (2-5%) and lower than Oriental populations 13-23%. A strong significant relation was found between major adverse cardiac events and carrying the variant allele CYP2C19 *2 (P = 0.001). On the other hand, there was no significant relation between major adverse cardiac events and carrying the variant allele CYP2C19 *3 (P = 0.324). Conclusion The CYP2C19 *2 allele is relatively common in our population, and its associated reduced metabolic activity deserves attention as it leads to an increased incidence of major adverse cardiac events in the follow-up of patients receiving clopidogrel.

HLA-DQ2 and -DQ8 haplotypes frequency and diagnostic utility in celiac disease patients of Gaza strip, Palestine.

PURPOSE: Celiac disease (CD) diagnosis can be established by serological and small bowel biopsy (SBB), while absence of HLA-DQ2 and -DQ8 haplotypes excludes the disease. The present study aims at evaluating the diagnosis of a representative sample of pediatric and adult CD patients of Gaza strip in light of DQ2 and DQ8 haplotypes expression. METHODS: Unrelated CD patients (n = 101) and matched healthy controls (n = 97) were genotyped for DQA1*05, DQB1*02 and DQB1*03:02 alleles by allele-specific real-time PCR. The diagnosis was re-evaluated according to the patient laboratory tests and HLA-DQ genotype. RESULTS: The diagnosis of 35 patients who have been managed for CD could not be confirmed. Twenty-five of them were diagnosed upon their clinical presentation only. The remaining were either negative for serological and SBB tests or negative for HLA-DQ haplotypes. The HLA-DQ alleles were negative in 4 SBB and one Anti-EMA positive patients. The frequency of DQ2 and DQ8 haplotypes among the remaining 65 confirmed cases was 70.8 and 15.4%, respectively, compared to 17.5 and 27.8% in the controls. The DQB1*02 allele was the most common in the cases (84.6%) followed by DQA1*05 allele (80%) and DQB1*03:02 allele (20%). The DQA1*05 allele was commonest in the control group (54.6%) followed by DQB1*02 allele (42.3%) and DQB1*03:02 allele (28.9%). CONCLUSIONS: Absence of HLA-DQ2 and HLA-DQ8 genotyping in the workup of patients may result in CD misdiagnosis, particularly in a setting with poor histopathological diagnostic capacity.

Homocysteine and vitamin B12 status and iron deficiency anemia in female university students from Gaza Strip, Palestine.

OBJECTIVE: Nutritional deficiencies are very significant to the overall health of humans at all ages and for both genders, yet in infants, children and women of childbearing age these deficiencies can seriously affect growth and development. The present work is aimed to assess homocysteine and vitamin B12 status in females with iron deficiency anemia from the Gaza Strip. METHODS: Venous blood samples were randomly collected from 240 female university students (18-22 years old) and parameters of the complete blood count, serum ferritin, homocysteine and vitamin B12 were measured. Statistical analysis included the t-test and analysis of variance (ANOVA) using the IBM SPSS software (version 18). Statistical significance was set for p-values <0.05. RESULTS: The results revealed that 20.4% of the students have iron deficiency anemia. The mean serum vitamin B12 level in females with iron deficiency anemia (212.9±62.8pg/mL) was significantly lower than in normal controls (286.9±57.1pg/mL) and subjects with microcytic anemia and normal ferritin (256.7±71.1pg/mL). Significantly higher serum homocysteine levels were reported in the iron deficiency anemia group (27.0±4.6µmol/L) compared to normal controls (15.5±2.9µmol/L) and in subjects with microcytic anemia and normal ferritin (18.1±2.7µmol/L). Statistically significant negative correlations were reported for serum homocysteine with serum ferritin, vitamin B12, hemoglobin, and hematocrit levels. CONCLUSION: Important associations were found between serum homocysteine and markers of iron deficiency. Monitoring homocysteine levels might be essential to understand the development of different clinical conditions including anemia. It seems necessary to conduct prospective trials to determine whether treating anemia ameliorates homocysteine levels.

Homocysteine and vitamin B 12 status and iron deficiency anemia in female university students from Gaza Strip, Palestine

OBJECTIVE: Nutritional deficiencies are very significant to the overall health of humans at all ages and for both genders, yet in infants, children and women of childbearing age these deficiencies can seriously affect growth and development. The present work is aimed to assess homocysteine and vitamin B12 status in females with iron deficiency anemia from the Gaza Strip. METHODS: Venous blood samples were randomly collected from 240 female university students (18-22 years old) and parameters of the complete blood count, serum ferritin, homocysteine and vitamin B12 were measured. Statistical analysis included the t-test and analysis of variance (ANOVA) using the IBM SPSS software (version 18). Statistical significance was set for p-values <0.05. RESULTS: The results revealed that 20.4% of the students have iron deficiency anemia. The mean serum vitamin B12 level in females with iron deficiency anemia (212.9 ± 62.8 pg/mL) was significantly lower than in normal controls (286.9 ± 57.1 pg/mL) and subjects with microcytic anemia and normal ferritin (256.7 ± 71.1 pg/mL). Significantly higher serum homocysteine levels were reported in the iron deficiency anemia group (27.0 ± 4.6 µmol/L) compared to normal controls (15.5 ± 2.9 µmol/L) and in subjects with microcytic anemia and normal ferritin (18.1 ± 2.7 µmol/L). Statistically significant negative correlations were reported for serum homocysteine with serum ferritin, vitamin B12, hemoglobin, and hematocrit levels. CONCLUSION: Important associations were found between serum homocysteine and markers of iron deficiency. Monitoring homocysteine levels might be essential to understand the development of different clinical conditions including anemia. It seems necessary to conduct prospective trials to determine whether treating anemia ameliorates homocysteine levels. .

Risk factors of cardiovascular disease among children with chronic kidney disease in Gaza strip.

BACKGROUND: Chronic kidney disease (CKD) is increasingly recognized as a global public health problem. Cardiovascular disease (CVD) is a major cause of mortality in patients with mild-to-moderate CKD and end-stage renal disease. There is accumulating evidence that the increase in CVD burden is present in CKD patients prior to dialysis, due both to conventional risk factors and kidney-specific disease. Detection and initiation of treatment for CVD risk factors at early stages of CKD should be effective in reducing CVD events before as well as after the onset of kidney failure. MATERIALS AND METHODS: The study sample consisted of a total of 112 subjects aged ≤12 years: 60 CKD patients and 52 healthy control individuals. All subjects were investigated for a group of CVD risk factors such as: Hypertension, diabetes, dyslipidemia, physical inactivity, body mass index (BMI), family history of CVD, hypoalbuminemia, albuminuria, anemia, Ca x P product, and inflammation in terms of C-reactive protein (CRP). RESULTS: Patients (40 males and 20 females) were categorized into four CKD stages (2, 3, 4, and 5) where, Stage 4 had the highest frequency, followed by Stages 3, 5 and 2. Evaluation of the patients indicated that they were shorter, had lower weight and had higher systolic and diastolic blood pressure as compared with control subjects. Frequency of physical inactivity among patients was two-fold higher than controls (50% vs. 25%). The patients showed significantly higher levels of cholesterol (163.6±39.8 vs. 141.8±24.2 mg/dL, P<0.0001), triglycerides (145.5±67.1 vs. 82.9±39.8 mg/dL, P<0.0001), low-density lipoprotein (92.6±31.9 vs. 72.5±19 mg/dL, P<0.0001) and albumin/creatinine ratio (1792±3183 vs. 11.1±6.6 mg/g, P<0.0001). Moreover, the patients had lower levels of high-density lipoprotein (41.9±11.0 vs. 52.7±11.7 mg/dL, P<0.0001), hemoglobin (9.8±1.4 vs. 11.9±0.8 g/dL, P<0.0001) and albumin (4.6±0.6 vs. 4.8±0.2 g/dL, P=0.012). The CRP showed higher occurrence among patients (40% were positive for CRP). Calcium and phosphorus evaluation showed significantly lower calcium and higher phosphorus among patients. However, the difference in Ca X P product was not statistically significant. CONCLUSIONS: The study indicates that many of the CVD risk factors are associated with the different stages of CKD in children patients prior to dialysis, and that some of these factors are exacerbated as CKD progresses.

Antidiabetic activity evaluation of glimepiride and Nerium oleander extract on insulin, glucose levels and some liver enzymes activities in experimental diabetic rat model.

The present study is aimed to assess the therapeutic potential of sulfonylurea drug glimepiride in comparison with Nerium oleander plant extract on insulin, glucose levels and some liver enzymes activities in streptozotocin-induced diabetic rats. Rats were rendered diabetic by intraperitoneal injection of a single dose of 50 mg kg(-1) body weight streptozotocin. Rats with serum glucose levels > 200 mg dL(-1) were subdivided into three sub-groups: the first sub-group were remained without treatment and considered as diabetics. The second and third subgroups were orally administered 0.1 mg kg(-1) body weight/day glimepiride and 250 mg kg(-1) body weight/day Nerium oleander, respectively for 4 weeks. Streptozotocin-induced diabetic rats showed hypoinsulinemia and hyperglycemia compared to controls. Strong negative correlation (r = -0.8) was found between serum insulin and glucose levels in diabetic rats. This correlation was +0.4 and -0.3 in glimepiride and Nerium olender-treated rats, respectively implying that glimepiride and plant extract improved insulin and glucose levels with the former was more efficient. The activities of serum aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase were significantly increased in streptozotocin-induced diabetic rats compared to controls. Treatment of diabetic rats with glimepiride or Nerium oleander extract also improved liver enzymes activities.